Herpes simplex virus is a useful model for studying the mechanisms involved in DNA replication in eukaryotic cells. Our current efforts are directed toward studying these processes with purified proteins. To this end, we have developed a transfection assay which has allowed us to identify all of the viral genes necessary for viral DNA replication. This system is based on results from several laboratories showing that transfected plasmid DNAs containing either of the two known origins of DNA replication, ORI-S and ORI-L are replicated in HSV-1 infected cells. We have found that a combination of five cloned restruction fragments of HSV-1 can supply all of the functions necessary for the replication of plasmids containing an HSV origin of replication. These five fragments are: XbaI C (0.0 - 0.294), XbaI F (0.294 - 0.453), XbaI E (0.453 - 0.641), XbaI D (0.641 - 0.830), and EcoRI JK (0.0 - 0.086; 0.830 - 0.865). We have now subcloned each of these fragments to precisely locate the essential genes, and have found a total of eight essential genes. This information will be used to purify each of the viral gene products involved in DNA replication, and the purified proteins will be used as a starting point to reconstruct DNA replication in vitro.